Neovascularization, vasculogenesis, and angiogenesis are terms which describe the process whereby new capillaries are formed.
The development of a vascular supply is essential for the growth, maturation, and maintenance of normal tissues. It is also required for wound healing and the rapid growth of solid tumors and is involved in a variety of other pathological conditions. Current concepts of angiogenesis, based in large part on studies on the vascularization of tumors, suggest that cells secrete angiogenic factors which induce endothelial cell migration, proliferation, and capillary formation. While the factors that induce anglogenesis in situ are not well identified, numerous factors have been identified which induce vessel formation in vitro or in vivo in animal models. These include: aFGF, bFGF, TGF-.alpha., TNF-.alpha., VPF, or VEGF, monobutyrin, angiotropin, angiogenin, hyaluronic acid degradation products, and AGE-products. Also, many compounds have been described as inhibitors of anglogenesis including a cartilage-derived inhibitor, identified as TIMP, PF-4, thrombospondin, laminin peptides, heparin/cortisone, minocycline, fumagillin, difluoromethyl ornithine, and sulfated chitin derivatives.
The major development of the vascular supply occurs during embryonic development, at ovulation during formation of the corpus luteum, and during wound and fracture healing. However, many pathological disease states are characterized by augmented angiogenesis including tumor growth, diabetic retinopathy, neovascular glaucoma, psoriasis, and theumatoid arthritis among other conditions. During these processes normally quiescent endothelial cells which line the blood vessels sprout from sites along the vessel, degrade extracellular matrix barriers, proliferate, and migrate to form new vessels. These processes are believed to be induced by factors secreted by the tissues to be vascularized and are often referred to as angiogenic factors. Angiogenic factors are secreted from surrounding tissue during the process which directs the endothelial cells to degrade stromal collagens, undergo directed migration (chemotaxis), proliferate, and reorganize into capillaries.
Several models for including angiogenesis in vivo have been developed. The corneal pocket assay involves the surgical implantation of polymer pellets containing angiogenic factors in the cornea of larger animals such as rabbits. Quantitation is difficult and the number of tests conducted is limited. The chick chorioallantoic membrane (CAM) assay involves the removal and transfer of a chick embryo from the shell to a cup. The angiogenic material is dried on a glass cover slip and placed on the chorioallantoic membrane and the appearance of new vessels is observed. The rabbit ear chamber assay requires the surgical insertion of a glass or plastic viewing device and measurement of capillary migration by microscopy. However, it is difficult to apply angiogenic materials in this assay. The rat dorsal air sac assay involves implants of stainless steel chambers containing angiogenic factors and is difficult to quantiate. An alginate assay which generates an angiogenic response has been described which involves the injection of tumor cells encased in alginate subcutaneously into mice. The accumulation of hemoglobin in the injected gel is used to quantitate the angiogenic response.
While each of these assays have many uses, some suffer from several major disadvantages which make their application to the development of anti-angiogenic agents difficult and cumbersome. First, some of these assays are not readily quantitated. The responses observed are often graded as positive and negative which results in poor reproducibility. Secondly, agents must be embedded in controlled release polymers, a process which may lead to inactivation of many biological activators or inhibitors of angiogenesis. Finally, the assays are cumbersome because they either employ larger, expensive animals or because the application of the test substances is tedious. Several in vitro assays of endothelial cell growth, migration, and capillary tube formation while useful in unlocking specific mechanisms in the angiogenic process are, of course, only initial screening methods for angiogenic or angiostatic substances. The final test must employ in vivo animal testing.